1. Field of the Invention
The present invention relates to a total reflection fluorescent microscope, which can perform fluorescent observation by total reflection illumination.
2. Description of the Related Art
In recent years, functions of biological cells have been vigorously analyzed. In the function analysis of the cells, attentions have been paid especially to a total reflection fluorescent microscope which acquires a total reflection fluorescent image from a cell membrane and its vicinity as a device for observing the function of the cell membrane.
Total reflection illumination which locally illuminates only a sample in the vicinity of a glass surface is used in the total reflection fluorescent microscope. In the total reflection illumination, an evanescent light is used which oozes to a sample side by about several hundreds of nanometers in a boundary surface between glass and sample, and a background noise (scattered light and the like) is remarkably low. Therefore, fluorescent observation of even a molecular of fluorescent dyestuff is possible by the total reflection fluorescent microscope.
Additionally, in general, in the total reflection fluorescent microscope, a laser light beam is used as a light source. A total reflection fluorescent microscope in which the laser light beam is introduced into an optical illumination system of the microscope via a glass fiber is described, for example, in Jpn. Pat. Appln. KOKAI Publication No. 2002-169097.
However, the laser light source which produces the laser light beam is expensive, and additionally a monochromatic light is produced. Therefore, for example, in order to cope with fluorescent reagents having various excitation wavelength characteristics, a plurality of laser light sources have to be prepared. Therefore, the total reflection fluorescent microscope becomes further expensive, and additionally a large occupying space is also required for installing a plurality of laser light sources.
To solve the problem, as described in Jpn. Pat. Appln. KOKAI Publication No. 2002-236258, a total reflection fluorescent microscope has been proposed in which inexpensive white light sources such as a mercury lamp and a xenon lamp are used instead of the laser light source. The total reflection fluorescent microscope according to the Jpn. Pat. Appln. KOKAI Publication No. 2002-236258 is configured as follows. A ring slit for transmitting a light beam in an annular form is disposed in the optical illumination system disposed on an optical path of the light emitted from the white laser light beam. Moreover, when an image of the ring slit is projected onto an emission pupil surface of an objective lens, an illuminative light is guided only to an orbicular total reflection region around an emission pupil of the objective lens. Moreover, total reflection is performed in a boundary surface between a specimen and cover glass to produce the evanescent light, and a fluorescent dyestuff is excited.